Improving the fluorescent probe acridonylalanine through a combination of theory and experiment

Article abstract of DOI:10.1002/poc.3813

Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior F?rster resonance energy transfer acceptor to Acd in a peptide cleavage assay and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives, which can be used in peptides and proteins.

Full text of DOI:10.1002/poc.3813

Received: 15 December 2017
DOI: 10.1002/poc.3813
Accepted: 27 December 2017
S P E C I A L I S S U E A R T I C L E
Improving the fluorescent probe acridonylalanine through a
combination of theory and experiment
1
1
1
1,2
Itthipol Sungwienwong | John J. Ferrie
| Joomyung V. Jun | Chunxiao Liu
|
1
3
4
1
Taylor M. Barrett | Zachary M. Hostetler | Naoya Ieda | Amara Hendricks |
1
3
1
5
Anand K. Muthusamy | Rahul M. Kohli | David M. Chenoweth | George A. Petersson |
1
E. James Petersson
1
Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
2
Department of Applied Chemistry, China Agricultural University, Beijing, China
3
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
4
Department of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya‐shi, Aichi, Japan
5
Temple University Institute for Computational Molecular Science, Philadelphia, PA, USA
Correspondence
E. James Petersson, Department of
Abstract
Chemistry, University of Pennsylvania,
Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluores-
cence spectroscopy, but it can potentially be improved by being made longer
wavelength or brighter. Here, we report the synthesis of Acd core derivatives
and their photophysical characterization. We also performed ab initio calcula-
tions of the absorption and emission spectra of Acd derivatives, which agree
well with experimental measurements. The amino acid aminoacridonylalanine
2
1
13 South 34th Street, Philadelphia, PA
9104, USA.
Email: ejpetersson@sas.upenn.edu
George A. Petersson, Temple University
Institute for Computational Molecular
Science, 1925 N. 12th Street, Philadelphia,
PA 19122, USA
(Aad) was synthesized in forms appropriate for genetic incorporation and
Email: gpetersson@temple.edu
Present Address
peptide synthesis. We show that Aad is a superior Förster resonance energy
transfer acceptor to Acd in a peptide cleavage assay and that Aad can be
activated by an aminoacyl tRNA synthetase for genetic incorporation. Together,
these results show that we can use computation to design enhanced Acd
derivatives, which can be used in peptides and proteins.
Amara Hendricks, Department of
Chemistry Oakwood University 7000
Adventist Blvd NW, Huntsville, AL 35896,
USA.
Anand K. Muthusamy, Department of
Biology California Institute of Technology
1
200 East California Boulevard, Pasadena,
KEYWORDS
CA 91125, USA.
Acridone, amino acid, electronic structure calculation, fluorescence spectroscopy
DEDICATION
that James was coming to Caltech, he was very proud of
the affirmation of George's appreciation of his graduate
George Petersson studied with John D. Roberts, earning
his PhD in 1970, and his son, James, studied with Dennis
Dougherty, earning his PhD in 2005. When Jack learned
education in Jack's group. The photo below of Jack,
George, James, and Dennis, taken during one of George's
visits with his son at Caltech, occupied a prominent place
Funding information: University of Pennsylvania; National Institutes of Health (NIH), Grant/Award Numbers: RR‐023444, RR‐022442 and GM‐071399;
National Science Foundation (NSF), Grant/Award Numbers: CHE‐1708759, MRI‐0820996, CHE‐1337449, MRI‐1625061 and DGE‐1321851; Royal Thai
Foundation; Japan Society for the Promotion of Science; Temple University; Gaussian Inc.
Itthipol Sungwienwong and John J. Ferrie contributed equally to this work.
J Phys Org Chem. 2018;e3813.
wileyonlinelibrary.com/journal/poc
Copyright © 2018 John Wiley & Sons, Ltd.
1 of 10
https://doi.org/10.1002/poc.3813

2
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SUNGWIENWONG ET AL.
in Jack's office. The connection between James and David
Chenoweth, friends and colleagues at the University of
Pennsylvania, was also forged at Caltech. David earned
his PhD with Peter Dervan in 2008 and was influenced
by Jack's many contributions to Physical Organic Chemis-
try, including his seminal work in benzyne chemistry.
Making the circle complete, Anand Muthusamy, who
worked on the acridone project as an undergraduate in
James' laboratory, began his PhD study at Caltech in
distances in the 15 to 40 Å range, and thioamide/Mcm
or thioamide/Acd electron transfer quenching pairs for
short distance (<15 Å) measurements.
[
6]
In addition to being better suited to short dis-
tance ranges than Fam or Tmr, Acd is small enough
to be directly genetically incorporated, rather than
posttranslationally attached, and is less likely to disrupt
[
7]
protein folding. This allows one to place a chromo-
phore on the interior of a protein and to label proteins
that cannot be reversibly unfolded and refolded (eg, our
2
016. George, James, and David are delighted to have this
[
8]
opportunity to recognize the impact that Jack Roberts had
on their lives.
published labeling of LexA). Having the chromophore
attached by a short sidechain rather than a Cys
maleimide or “click” chemistry triazole linker also
reduces the positional uncertainty of the FRET probe
with respect to the protein backbone. Thus, distance mea-
surements from FRET should more reliably report on
changes in protein conformation. We and others have
previously shown that Acd can be a valuable probe for
3
protein study because of its small size (222 Å ), high
quantum yield in water (ϕ = 0.95), unusually long fluo-
[9]
rescence lifetime (τ ~15 ns), and high photostability.
We have developed an engineered aminoacyl tRNA syn-
thetase (RS)/ orthogonal tRNA pair for selective Acd
9b
incorporation by unnatural mutagenesis.
This has
allowed us to label proteins and peptides with
methoxycoumarin/Acd FRET pairs either through Mcm
incorporation by solid‐phase peptide synthesis or by
attachment of methoxycoumarin‐maleimide to a Cys res-
idue in a protein. While many aspects of the Mcm/Acd
FRET pair are optimal, such as significant spectral over-
lap and a high Mcm extinction coefficient at 325 nm
where Acd has a minimum, one disadvantage is the small
Acd Stokes shift, which leads to significant overlap of
their emission spectra. This overlap necessitates a chal-
lenging deconvolution of the Mcm/Acd spectra in order
to determine FRET efficiencies and distance measure-
ments. Thus, an Acd derivative with a larger Stokes shift
would be desirable for Mcm FRET.
1
| INTRODUCTION
6b
Fluorescence spectroscopy can be a valuable tool for
studying the structural dynamics of proteins and
protein/protein interactions. There are several common
types of protein experiments that use fluorescence spec-
troscopy: folding/conformational change experiments,
[1]
[
2]
binding experiments, and proteolysis experiments
Figure 1). Changes in fluorescence can be used to track
(
protein structural change on the nanosecond timescale
using distance‐dependent chromophore interactions:
either Förster resonance energy transfer (FRET) or
quenching by photo‐induced electron transfer). Such
studies require fluorescent probes that enable accurate
measurement on a variety of distance ranges between
the two chromophores. FRET ranges are characterized
by the Förster radius, R , the distance of half‐maximal
energy transfer for any chromophore pair.
example, the common FRET pair fluorescein (Fam)/
[
3]
In addition to improving Mcm FRET, we also wish to
alter other fluorescent properties of Acd, such as red‐
shifting excitation and emission, increasing the extinction
coefficient, and altering solvatochromic effects to make
brighter derivatives that are better suited to microscopy
or single‐molecule fluorescence applications. Sisido and
0
[
4]
For
[
10]
coworkers
have previously shown that some of these
tetramethylrhodamine (Tmr) has an R of 47 Å and is
useful to measure distances in the 30 to 90 Å range.
effects can be achieved by simply homoligating Acd with
a benzene ring (benzoacridonylalanine or Bad, 3)
to extend the π‐system. However, we wish to make
derivatives that can still be incorporated by the ribosome,
and Sisido's laboratory also showed that Bad was not
incorporable during in vitro translation with chemically
charged tRNA. Therefore, we will use crystal structures
and computational models of our evolved AcdRS and
0
[
5]
Since many interresidue distances in proteins are shorter
than this, one needs to complement the Fam/Tmr FRET
pair with other probe pairs that are better suited to shorter
interactions. With this in mind, the Petersson laboratory
has developed a methoxycoumarinylalanine (Mcm, 1)/
acridonylalanine (Acd, 2) FRET pair for monitoring

SUNGWIENWONG ET AL.
3 of 10
FIGURE 1 Förster resonance energy
transfer (FRET) experiments and
fluorescent amino acids. Top, protein
conformational changes, protein‐protein
interactions, and proteolytic cleavage can
be monitored by changes in the
intramolecular or intermolecular distance
between 2 FRET probes. Bottom,
fluorescent amino acids based on 7‐
methoxycoumarin and acridone cores
“
rules” for ribosomal permissivity established in previous
acridone literature. Finally, we synthesize Aad and
perform initial trials toward its genetic incorporation.
[7,11]
in vitro translation studies
to restrict Acd substitu-
tions to positions that will allow in vivo tRNA charging
and incorporation into proteins.
2
| RESULTS AND DISCUSSION:
Identifying Acd derivatives by making amino acid
analogs is synthetically laborious and unnecessary given
that Acd and Bad spectroscopic properties are identical
ACRIDONE DERIVATIVE
SYNTHESES
9a,
to the properties of the respective chromophore cores.
To synthesize acridone cores that were not commercially
available, we used 2 general strategies, functionalization
of acridone and cross‐coupling of prefunctionalized aryl
units. Nitration of acridin‐9(10H)‐one 5 was performed
with HNO in CH COOH at 50°C, giving a mixture 2‐
nitroacridin‐9(10H)‐one (6) and 4‐nitroacridin‐9(10H)‐
one (7) (variations in reaction conditions produced a
74% yield of 6 or 38% yield of 7, see Table S1 and
c, 10
Thus, to improve Acd fluorescence, we set out to
make a series of acridone (5) core derivatives in order
to identify derivatives with sufficiently improved
properties to warrant synthesis of the amino acid form
for incorporation into peptides. Previous studies of
acridone derivatives have shown that many of the
spectroscopic properties can easily be modulated through
substituent effects, providing strong precedent for our
3
3
[
13]
Figure S1).
Reduction of 6 and 7 using Na S and
2
[10,12]
work.
Moreover, focusing on the acridone core
NaOH in refluxing EtOH/water gave 2‐aminoacridin‐
9(10H)‐one (8) and 2‐aminoacridin‐9(10H)‐one (9),
respectively, in quantitative yields (Na S used for small
scale, Pd/C also used to reduce 7, see Figure S2 for
details)
We also used Buchwald‐Hartwig cross‐coupling
amination between methyl 2‐aminobenzoate (10) and aryl
bromides 11‐12, followed by Friedel‐Crafts cyclization, to
generate several acridone derivatives.
was accomplished using Pd(OAc)2, racemic 2,2′‐
bis(diphenyl‐phosphino)‐1,1′‐binaphthyl (rac‐BINAP),
and Cs CO to generate methyl 2‐(phenylamino)benzoates
makes computational modeling more tractable, with
the potential to further narrow the scope of synthetic
work by predicting absorption and emission spectra.
Here, we prepare a series of acridone derivatives
in order to validate the accuracy of our electronic
2
[
13]
(Scheme 1).
structure calculations and identify
a
derivative,
aminoacridonylalanine (Aad, 4) with substantially red‐
shifted emission. In addition, our calculations help to
explain the origin of fine structure in acridone spectra,
an explanation that is in conflict with the conclusions of
previous computational and experimental Acd spectros-
copy studies but is consistent with the larger body of
[
14]
Cross‐coupling
2
3
13 and 14 in 86% and 78% yield, respectively. Then, the

4
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SUNGWIENWONG ET AL.
SCHEME 1 Synthesis of nitroacridones
(
6 and 7) and aminoacridones (8 and 9)
acridone cores were achieved by saponification with
LiOH, followed by Friedel‐Crafts cyclization, to give 4‐
summarized in Figure 2 and Table 1, and the spectra of
each compound are reported separately in the
Supporting Information. The absorption and emission
spectra are normalized in Figure 2 for clarity, raw spectra
are shown in the Supporting Information, and extinction
coefficients and emission intensities are given in Table 1.
Since we ultimately endeavor to utilize these derivatives
as fluorescent unnatural amino acids, we attempted to
perform the spectroscopic characterization in phos-
phate‐buffered saline, pH 7.4. However, because of the
relatively low solubility of the benzoacridone compounds
in water, all measurements were performed in 1:1 aceto-
nitrile/phosphate‐buffered saline. Extinction coefficients
were obtained through serial dilutions of stocks of
300 μM, which is approximately the solubility limit of
benzoacridone in acetonitrile. Fluorescence emission
spectra were acquired under the same solvent conditions.
The parent acridone (5) absorbance spectrum features
2 major peaks in the near UV region with maxima at 382
and 398 nm as well as additional features below 300 nm.
Derivatization of the acridone core resulted in changes
of the absorbance profile that were both functional group
and position dependent. For example, introducing a
methoxy group in the 2 position in S1 resulted in an
approximately 15‐nm redshift in the absorbance
maximum, with minimal modulation of the line shape,
while introduction at the 4 position in 16 resulted in a
very minimal shift, with the multipeak profile becoming
less defined. Amino modification at either the 2 or 4
positions produces a singular broad feature that is signifi-
cantly redshifted. Moreover, the absorbance profile of
fluoroacridin‐9(10H)‐one
(15,
93%)
and
4‐
methoxyacridin‐9(10H)‐one (16, 96%) (Scheme 2).
The same strategy was used to form benzoacridone
derivatives. Buchwald‐Hartwig cross‐coupling with
methyl 3‐amino‐2‐naphthoate (17) and aryl bromides
(
11‐12 and bromobenzene) gave the corresponding
methyl 3‐(phenylamino)‐2‐naphthoates in 97‐98% yields
see Supporting Information). Then, the benzo[b]acridin‐
2(5H)‐one derivatives (18‐20) were formed by hydrolysis
(
1
and Friedel‐Crafts ring‐closing reactions in 85% to 89%
yields (Scheme 2).
These methods provided access to many acridone
derivatives for this study and are sufficiently general to
provide access to most desired derivatives for future
studies. In particular, the cross‐coupling route allows us
to access derivatives, such as 3‐aminoacridone, that may
be difficult to synthesize using nucleophilic aromatic
substitution, as the directing effects of the ortho carbonyl
and amino groups on each benzene ring in the acridone
core would strongly favor reactions at the 2 or 4 positions.
3
| RESULTS AND DISCUSSION:
SPECTROSCOPIC
CHARACTERIZATION
Following production of the various Acd derivatives, we
determined the absorbance and emission profiles, as well
as the extinction coefficient, of each core. These data are
SCHEME 2 Synthesis of acridones 15
and 16 and benzoacridones 18‐20

SUNGWIENWONG ET AL.
5 of 10
FIGURE 2 Absorption and emission
spectra of acridone derivatives. Spectra
determined in 1:1 acetonitrile/phosphate‐
buffered saline, pH 7.4. Spectra are shown
normalized to enable comparison of
changes in absorption and emission
maxima. Raw spectra are shown in the
Supporting Information
2
‐aminoacridone (8) is pH sensitive (see Figure S5). The
that both the unmodified and the 4‐amino Acd chromo-
phores are pH insensitive (see Figures S4 and S6). Lastly,
we observe that extension of the conjugated system in the
case of benzoacridone (18) results in an expected shift of
the absorbance to higher wavelengths but displays an
unexpected reduction in the extinction coefficient.
When modified with either fluoro (19) or O‐methyl (20)
substituents, the absorbance profile is minimally shifted,
and the peaks become less well‐resolved, similar to what
was observed when modifying the parent acridone
scaffold (15 and 16).
spectrum takes on a single broad absorbance feature very
similar to that of 4‐aminoacridone (9) at high pH, which is
dramatically reduced at low pH. It is important to note
TABLE 1 Calculated and observed photophysical parameters of
acridone derivatives
a
Calculated
Observed
b
c
b
c
Compound
λ
ex/ε
λ
em/Int.
λ
ex/ε
λem/Int.
Acd 5
389/2.43
408/1.87
464/2.06
419/0.47
400/0.61
365/1.19
400/0.95
375/0.62
446/0.81
446/0.61
456/0.67
411/1.00
…
398/0.70
402/0.37
434/0.60
420/0.52
400/0.59
378/0.51
396/0.82
384/0.69
438/0.58
448/0.54
454/0.61
412/1.00
…
Acridone substitution also elicited changes in the
emission profile and Stokes shift. The unmodified
acridone emission features 2 major peaks at 412 and
2
4
2
4
4
2
4
‐NO
2
2
6
7
8
9
‐NO
…
…
‐NH
2
2
508/0.21
492/0.59
397/0.48
444/0.47
421/0.35
495/0.31
496/0.24
512/0.25
527/0.16
540/0.01
412/0.65
447/0.63
431/0.37
507/0.42
508/0.21
512/0.14
435 nm with a minor peak around 460 nm. As in the
‐NH
absorbance profiles, the maxima of these peaks move to
lower energy upon modification with the O‐methyl
group (Table S1 or 16) with the multipeak profile becom-
ing less defined. Similarly, introduction of the amine
functionality resulted in a significant redshift as well as
a reduction of the multipeak line shape to a single broad
emission. Both compounds 8 and 9 exhibited a dramatic
increase in Stokes shift of approximately 100 nm
compared to the acridone core. Both also displayed a
sensitivity to pH, manifested as a decrease in emission
intensity with decreasing pH, compared to the unmodi-
fied core whose emission is insensitive to pH changes
in the 2 to 10 range. Finally, the benzo‐modified
‐F 15
‐OMe S1
‐OMe 16
Bz 18
4
‐F‐Bz 19
4
‐OMe‐Bz 20
a
Lowest energy absorption and highest intensity emission data reported here.
A full list of absorption and emission peaks is found in Table S2.
b
4
‐1
‐1
Extinction coefficients (ε) reported as 10 M cm .
c
Emission intensity (Int.) normalized the intensity of the highest emission
peak of acridone for both calculated and observed spectra.

6
of 10
SUNGWIENWONG ET AL.
acridone analogs all feature a nearly identical emission
profile approximately 100‐nm redshifted from the emis-
sion of the parent acridone compound. The combination
of benzo and fluoro or methoxy substitution further
redshifts the absorption spectrum but does not apprecia-
bly change the emission spectrum.
coefficients is excellent for the n → π* transition, the
calculations overestimate the intensity of the π → π*
absorption. However, what is most important is that
throughout the series of acridone derivatives, the
wavelengths and intensities of the π → π* absorption
and emission track very well with the experimental data,
showing that the calculations can clearly predict
acridone derivative properties to guide synthetic efforts
4
| RESULTS AND DISCUSSION:
ELECTRONIC STRUCTURE
CALCULATIONS
(
see Tables 1 and S2 and Figures S4‐S9).
For acridone, we performed more sophisticated
calculations, combining Franck‐Condon integrals from
vibrational calculations to generate spectra representing
acridone absorption and emission spectra in aqueous
solution. Bonding in excited states is generally not as strong
as in ground states, which reduces the zero point energy
(ZPE) from molecular vibrations. For example, the ZPE
for the lowest‐lying π → π* excited state of acridone
(115.76 kcal/mol) is 2.21 kcal/mol less than the ZPE for
the ground state (117.97 kcal/mol). This reduces the vertical
excitation energy to 75.39 kcal/mol (λex = 379 nm). The
electronically excited state then relaxes through internal
conversion to its vibrational ground state, lowering the
energy by 1.43 kcal/mol. This is followed by solvent relaxa-
tion, which lowers the energy of the excited state by an
additional 1.02 kcal/mol and raises the ground state energy
by 0.94 kcal/mol (Figure 4). The vertical emission
(69.64 kcal/mol or λem = 411 nm) is followed by relaxation
through internal conversion to the vibrational ground state,
lowering the energy by 2.08 kcal/mol. Solvent relaxation
then lowers the energy of the ground state by an additional
1.53 kcal/mol. We found that these Franck‐Condon
To understand the nature of the substituent effects on
acridone fluorescence, we performed ab initio electronic
structure calculations of the absorption and emission
spectra for all of the core derivatives. The calculations
used the APF‐D density functional as implemented
in the Gaussian16™ suite of programs with the
6
‐311+G(2d,p) basis set, which has been recommended
[15]
for calculations of fluorescence spectra.
The close
agreement between the calculated (Calc.) and experi-
mental (Obs.) spectra for the parent species, acridone,
clearly justifies this choice (Figure 3). For microscopy
and spectroscopy applications, the useful transition is
not the expected intense C═O n → π* excitation at
2
53/255 nm (Calc./Obs.). Rather, it is the weaker
π → π* excitation with peaks at 370/376 nm (Calc./
Obs.) and 389/398 nm (Calc./Obs.). The emission
spectrum shows an approximately 10‐nm Stokes shift
with π* → π emission peaks at 411/412 nm (Calc./Obs.)
and 434/436 nm (Calc./Obs.). We note that although
the agreement of the calculated and observed extinction
FIGURE 3 Calculated acridone spectra. Top left, experimental and calculated, Franck‐Condon corrected spectra, with transitions used to
determine the calculated spectrum shown as vertical bars. Bottom left, primary molecular orbitals (MOs) involved in the n → π* (approximately
2
50 nm) and π → π* (approximately 380 nm) transitions of acridone. Right, Jablonski diagram showing strategy for using frozen ground or
excited state solvation in geometry optimization and frequency calculations to determine electronic spectra

SUNGWIENWONG ET AL.
7 of 10
FIGURE 4 Aad is a superior Förster
resonance energy transfer (FRET) acceptor
for Mcm. Top, absorbance (solid lines) and
fluorescence emission (dashed lines) of
Mcm (purple), Acd (blue), and Aad
(
orange) with spectral overlap shaded.
Both Acd and Aad have low absorbance at
the excitation maximum of Mcm (325 nm)
and comparable spectral overlap (shaded
areas), but Aad emission can more easily
be distinguished from Mcm emission.
Bottom left, normalized changes in FRET
ratio (380:480 nm for Acd, 380:530 nm for
Aad) of 5 μM Xxx‐Leu‐Leu‐Lys‐Ala‐Ala‐
Ala‐Mcm (Xxx = Acd or Aad) peptides
upon rapid mixing with 1 μM trypsin
protease. Data points from 3 trials shown
to demonstrate reproducibility, with
average shown as a smooth curve. Bottom
right, fluorescence emission spectra, with
excitation at 325 nm, of Acd or Aad
peptides before and after proteolysis.
Spectra normalized based on area under
the curve
corrected spectra have excellent agreement with the
observed acridone spectra, particularly for the features in
the π → π* absorption and emission peaks. We note that
previous computational studies of acridone by Oshima,
accompanied by gas phase electronic spectra measure-
ments, concluded that the various vibronic modes that
they observed for the π → π* absorption and emission were
as any of the benzo derivatives, but its smaller size and
greater polarity make it more favorable for eventual
genetic incorporation. While the absorption and emission
spectra of 2‐ and 4‐aminoacridone are relatively similar,
the 2‐amino isomer was chosen because it is brighter,
and our modeling studies (below) suggest that it is likely
to be genetically incorporable. Since the 2‐aminoacridone
core shows a larger Stokes shift than the acridone core
with comparable spectral overlap and selective excitability
(ie, the maximum in Mcm absorbance coincides with a
minimum in Acd or Aad absorbance), it can function as
a superior FRET partner for Mcm. Our spectral overlap
calculations indicate that acridonylalanine (Acd, 2) and
[16]
the result of different hydration clusters.
While this
explanation may be valid for gas phase measurements,
solution‐phase measurements show a similar spectral
shape for acridone absorption and emission in polar,
hydrogen‐bonding solvents like water, and in nonpolar
9c
solvents like THF. Thus, the explanation arising from
our Franck‐Condon corrected calculations (determined
with continuum solvation), that the multiple peaks at
approximately 380 nm arise from internal acridone vibra-
tional modes, seems to fit better with solution phase data.
aminoacridonylalanine (Aad, 4) have similar R values
0
(Acd: 25.3 Å, Aad: 26.5 Å), but the emission of Aad at
530 nm will be easier to monitor in ratiometric FRET
experiments, and no deconvolutions with Mcm emission
will be necessary (Figure 4). Although 2‐aminoacridone
is commonly used in glycan experiments, and acyl
2‐aminoacidone peptides have been used previously in
protease sensors, to our knowledge, amino acid forms
such as Aad have not been made previously or used in
5
| RESULTS AND DISCUSSION:
SYNTHESIS OF
AMINOACRIDONYLALANINE FOR
PROTEIN STUDIES
12b, 17
FRET applications.
Acridonylalanine (2) was synthesized as we have
previously reported. Nitration of Acd was conducted
similarly to nitration of acridone, affording a single nitro
We chose to generate the amino acid form of 2‐
aminoacridone (8) since its emission was as redshifted

8
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SUNGWIENWONG ET AL.
FIGURE 5 Aad activation by AcdRS1.
Left, Acd and Aad adenylation measured
i
based on inorganic pyrophosphate (PP )
formation in an enzyme‐coupled Malachite
green assay. Control experiments lacking
adenosine triphosphate (ATP) show that
the low levels of Aad activity are
significant. Right: Aad docked in the
AcdRS1 active site by alignment with Acd
in an energy‐minimized model generated
20b
from AcdRS1 PDB coordinates 4PBR.
see ESI for details
[
8]
isomer in 39% isolated yield (>90% conversion based on
integration of the chromatogram). The nitration reaction
mixture was basified and sodium sulfide added to
perform the reduction to give Aad (4) in 86% yield
AcdRS1, although less efficiently than Acd (Figure 5).
These results can be understood with a simple model in
which Aad is aligned with Acd in our published AcdRS1
docking model, based on the crystal structure of AcdRS1
8, 20b
(see Supporting Information). The position of the amino
with Brb.
As one can see in Figure 5, the Aad exocyclic
group was confirmed by NMR and by comparison of the
absorption and emission spectra to the spectra of 2‐ and
amine makes contact with the sidechains of Val164 and
Ala167. Thus, mutation of these residues to smaller or
more polar residues may improve Aad charging. Generat-
ing a library of randomized mutants of the 164 to 167 loop,
followed by selection of the most active clones, is also
likely to result in an efficient AadRS for in vivo incorpora-
tion of Aad.
4‐aminoacridone (8 and 9). Protected forms of Acd
and Aad (see Figure S3) were generated for solid‐phase
peptide synthesis, and the peptides Xxx‐Leu‐Leu‐Lys‐
Ala‐Ala‐Ala‐Mcm (Xxx = Acd or Aad) were synthesized.
The peptide sequence was chosen as a protease substrate
based on previous studies in our laboratory so that
[18]
cleavage should result in a decrease in FRET.
Cleav-
age by the proteases trypsin and cathepsin B was tested
by exciting at 325 nm and monitoring emission at
6 | CONCLUSIONS
3
(
80 nm (Mcm) and either 480 nm (Acd) or 530 nm
Aad). After confirming that trypsin proteolysis
proceeded at a reasonable rate in a multiwell plate assay
see Figures S10‐S11), we measured cleavage rates in a
We can draw several conclusions from our results.
Firstly, as anticipated from previous literature reports,
we are able to modulate the fluorescence of the acridone
core through relatively simple substitutions. These
substitutions, introduced through direct modification of
acridone or through cross‐coupling and cyclization, are
compatible with eventual usage in generating Acd
amino acid derivatives. Secondly, the close correlation
(
stopped‐flow fluorometer. As one can see in Figure 4,
while both peptides give usable data, the change in
FRET ratio is twofold greater for the Aad peptide than
the Acd peptide.
Site‐specific genetic incorporation of Aad requires a
tRNA synthetase, or RS, that efficiently and selectively
adenylates Aad and then charges an orthogonal tRNA
(% differences λ : 2.35, λem: 2.40) between our
ex
calculated and observed absorption and emission spectra
gives us confidence that we can predict the spectra for
acridone derivatives, providing guidance for future
synthetic efforts. This will be particularly valuable for
targeting multiply‐substituted derivatives, where the
number of possibilities is geometrically larger and
the synthesis will be more challenging. Finally, Aad
appears to be superior to Acd for several fluorescence
applications. As demonstrated here, the redshifted
emission of Aad provides superior dynamic range in
ratiometric FRET measurements with an Mcm donor.
Furthermore, its absorption and emission wavelengths
[19]
with this activated Aad ester.
As a first step toward
achieving an AadRS, we tested Acd and Aad adenylation
by AcdRS1 (also referred to as clone G2), a permissive
Methanocaldococcus jannaschii TyrRS derivative that
incorporates Acd and also incorporates N‐phenyl p‐
aminophenylanine (Npf) and 4‐(2′‐bromoisobutyramido)
8, 9b, 20
phenylalanine (Brb).
Using a purified form of
AcdRS1 in a Malachite green assay that we have previously
used to characterize the relative activity of the enzyme
toward Acd and Npf, we find that Aad is activated by

SUNGWIENWONG ET AL.
9 of 10
make Aad amenable to use in microscopy applications
using typical filter sets for cyan fluorescent protein
excitation and yellow fluorescent protein emission.
Aad can also serve as a FRET donor to red wavelength
dyes such as indodicarbocyanine or sulforhodamine
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Using the strategy outlined here, we can
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ration of Acd derivatives. Our initial in vitro activity
results for Aad are very promising, and computational
models based on the crystal structure of AcdRS1 indi-
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with just 1 or 2 mutations. These rationally designed
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an AadRS, this would enable the furthest redshifted
fluorescent amino acid to be genetically incorporated
in living cells.
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This work was supported by the University of Pennsyl-
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include MALDI MS (NSF, MRI‐0820996); LCMS
and HRMS (NIH, RR‐023444); stopped flow fluorometer
(NSF, CHE‐1337449); and NMR (NIH, RR‐022442).
We thank the following for fellowship support:
Royal Thai Foundation (IS), NSF Graduate Research
Fellowship Program (DGE‐1321851, JJF), NIH
Chemistry Biology Interface Training Program (T32
GM071399, TMB and ZMH), and the Japan Society for
the Promotion of Science (NI). The computational
component of this research was supported by Temple
University, Gaussian, Inc., and by the NSF (MRI‐
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1
625061)
ORCID
John J. Ferrie http://orcid.org/0000-0001-7934-7266
E. James Petersson http://orcid.org/0000-0003-3854-9210
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How to cite this article: Sungwienwong I, Ferrie
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acridonylalanine through a combination of theory
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